Most cancer cells share two abnormalities: uncontrolled proliferation and suppressed apoptosis. A direct relationship has been shown between the expression level of nucleophosmin (NPM) and a variety of human cancers including leukemia evolved from Fanconi anemia (FA), a genetic disorder associated with bone marrow failure, clonal proliferation of hematopoietic cells, and transformation to acute leukemia. This suggests a potential role of NPM in cancer development. We identified NPM as a novel cellular inhibitor of the pro-apoptotic protein kinase PKR, and demonstrated that NPM regulates PKR activity and suppresses apoptosis induced by mitogenic inhibitors and therapeutic agents in hematopoietic cells of FA. Our preliminary studies also indicated that NPM is a component of the so-called FA-PKR complex, which functions as a signaling intermediate in cellular responses to apoptotic and survival signals. We hypothesize that NPM suppresses apoptosis and promotes proliferation through inhibiting the pro-apoptotic kinase PKR and accelerating cell-cycle progression and that leukemic evolution in FA and other cancer-prone bone marrow diseases may require increased expression of NPM to maintain survival and proliferation. The long term goals of our study are to utilize FA as an experimental system to examine the role of NPM in hematopoiesis and leukemic evolution. We plan to build on our preliminary characterizations of NPM-PKR interaction and NPM deregulation during FA disease progression to explore in molecular details the biochemical and functional alterations of NPM in the context of NPM expression, NPM-PKR interaction, the integrity of the FA-PKR complex, PKR activity, and apoptosis in BM cells from normal donors and FA patients at different stages of disease progression. We will also investigate whether regulation of PKR by NPM and the FA-PKR protein complex are linked mechanistically in these primary BM cells. We will employ three unique FA cell lines derived from FA patients at different stages of the disease progression to study the mechanism by which NPM promotes proliferation and suppresses apoptosis. To study the role of NPM in clonal proliferation, we will determine effects of NPM expression on proliferation and differentiation of FA evolved leukemic cells and hematopoietic stem/progenitor cells of FA mice. The proposed study will be of fundamental importance in understanding the evolution of bone marrow failure syndromes such as Fanconi anemia to clonal hematologic diseases such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In addition, study of biological consequences of NPM alterations in the context of PKR suppression during FA disease progression may provide rationale for exploiting this molecule for detection and prevention of certain cancers including FA-evolved leukemia.